Several publications and patent documents are referenced in this application by author name, year and journal of publication or number in parentheses in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications is incorporated by reference herein.
Thrombin is the final protease in the blood coagulation cascade, and converts soluble fibrinogen to an insoluble fibrin clot. Fibrin/fibrinogen interacts with thrombin in several distinct ways. First, fibrinogen serves as a substrate for thrombin, in which thrombin cleaves short fibrinopeptides of 16 and 14 amino acids, respectively, from the amino termini of the α and β chains of fibrinogen (1). In addition, thrombin can also bind to fibrin at sites that are distinct from thrombin's catalytic site (2). Fibrin contains both high and low affinity sites for thrombin (3). A low affinity site has been mapped to the central E domain of fibrin (4), and involves residues α23-51 (5) and the newly-exposed amino terminus residues β15-42 (3). These low affinity interactions are believed to facilitate thrombin cleavage of the fibrinopeptides. In addition to the low affinity thrombin binding sites, fibrin also contains high affinity binding sites. Studies by Meh et al. (3, 6) showed that a high affinity thrombin binding site is located on the γ′ chain of fibrinogen.
The γ′ (or γB) chain arises from alternative processing of the γ chain mRNA (7, 8), and constitutes about 7% of the total γ chains in fibrinogen (9). The γ′ chain carboxyl terminus is highly anionic, with seven Glu/Asp residues within the last seventeen amino acids, and contains tyrosine O-sulfate residues (6, 10). γ′-chain containing fibrinogen consists primarily of a heterodimer with one γ′ chain and one γA chain, while the more common form of fibrinogen contains two γA chains (11, 12). Earlier studies on thrombin binding to fibrin fragments (4) showed no binding to the D domain of fibrinogen where the γ′ chain resides. However, the carboxyl terminus of the γ′ chain is cleaved by plasmin during fibrinolysis (13), which may explain the lack of thrombin binding seen in these earlier studies with fragment D.
Binding of thrombin to these sites has important physiologic consequences. Clot-bound thrombin is resistant to inactivation by its natural plasma inhibitor, ATIII, even in the presence of the anticoagulant glycosaminoglycan, heparin (14, 15). In the absence of fibrin, heparin increases the rate of thrombin inactivation by ATIII by forming a ternary-complex with thrombin and ATIII (16). Fibrin also increases thrombin's amidolytic and proteolytic activities (17). In addition, in vitro data shows that clots made from γA/γ′ fibrinogen are resistant to fibrinolysis by plasmin. This may be due, at least in part, to increased crosslinking by factor XIIIa (18), a plasma transglutaminase that is activated by thrombin. Factor XIII is activated more rapidly in the presence of γA/γ fibrin than γA/γA fibrin (17).
The binding site on thrombin for the γ′ chain has been the focus of intense research. Two potential binding sites for the highly negatively-charged γ′ chain include anion-binding exosites I and II (19). Exosite I binds to fibrinogen near the amino terminus to facilitate fibrinopeptide cleavage (20), and can bind to heparin cofactor II (21) or to the thrombin receptor (22) as well. The leech salivary anticoagulant protein, hirudin, also binds to exosite I and prevents fibrinogen binding (23). In contrast to exosite I, exosite II is often considered to be a glycosaminoglycan binding site that mediates heparin-accelerated inhibition by ATIII (24, 25), and binds chondroitin sulfate residues in thrombomodulin (26). However, exosite II also binds a proteinaceous ligand, the platelet cell-surface thrombin receptor, GPIbα (27).
The above-mentioned studies demonstrate that thrombin, fibrinogen and fragments thereof are involved in a myriad of pathways critical for maintaining the quality and duration of the blood clotting reaction. Reagents which specifically modulate the activity of these proteins are desirable for the treatment of pathological disorders associated with aberrant clotting activity, such as thrombosis.